Influenza virus remains an unmet health issue. Although the virus is controlled with annual vaccines, these exhibit variable efficacy, and slow vaccine production means vaccines are not available during an emerging pandemic. Antivirals can stunt an influenza pandemic, but antivirals become ineffective over time due to selection of resistant viruses. These facts highlight the need to identify novel targets for development of new antiviral therapies to combat emerging influenza, control influenza-related disease, and enhance human health. There is urgent need to discover new molecular targets and produce novel antiviral therapies against influenza virus to prevent an emerging pandemic. Our long-term goal is to discover multiple targets that can be exploited in combination to block influenza viruses. Our objective is to characterize influenza nucleoprotein (NP) interactions essential for influenza RNA expression. NP is an essential component of the functional viral ribonucleoprotein (vRNP), responsible for viral mRNA transcription and RNA genome replication. Recently the FDA approved the first antiviral to target this influenza-virus complex. XOFLUZA is an endonuclease inhibitor targeting influenza PA protein of the vRNP complex and has great promise to inhibit influenza infection rapidly. The activity targeted in this new antiviral was discovered via publicly funded research nearly 40 years ago (Krug, 1981) and exemplifies the importance of our proposed research. Our work is complementary and focuses on the nucleoprotein of the vRNP. Our central hypothesis is that elucidating molecular interactions essential for influenza RNA expression will identify novel antiviral targets. It is reasoned that targeting a highly conserved viral protein is less likely to result in evolution of resistance, particularly when the conserved target is attacked at multiple sites. Once better understood, interactions essential to viral RNA expression will reveal novel ways of preventing an emerging influenza virus pandemic. To test our central hypothesis and attain our overall objective, we will pursue the following specific aims: 1) Characterize influenza nucleoprotein interactions essential for vRNP function and 2) Define influenza nucleoprotein interaction with non- structural protein 1. The proposed research is significant because we will identify multiple antiviral targets within conserved regions of NP, reasoned to be less prone to the development of resistance. The proposed research is innovative because it will identify novel targets to inhibit influenza, possibly including host factors that interact with NP but serve redundant functions for the host. This research will advance knowledge of the physiology of influenza virus and provide fundamental information that can be harnessed to support development of new anti-influenza therapies and enhance human health.